Effects of the dietary linoleic acid to linolenic acid ratio for Nile tilapia (Oreochromis niloticus) breeding females

Journal Pre-proof Effects of the dietary linoleic acid to linolenic acid ratio for Nile tilapia (Oreochromis niloticus) breeding females Tamira Maria Orlando, Táfanie Valácio Fontes, Renan Rosa Paulino, Luis David Solis Murgas, Jose Fernando López-Olmeda, Priscila Vieira Rosa PII:

S0044-8486(19)31317-1

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734625

Reference:

AQUA 734625

To appear in:

Aquaculture

Received Date: 6 June 2019 Revised Date:

14 October 2019

Accepted Date: 21 October 2019

Please cite this article as: Orlando, T.M., Fontes, Tá.Valá., Paulino, R.R., Solis Murgas, L.D., López-Olmeda, J.F., Rosa, P.V., Effects of the dietary linoleic acid to linolenic acid ratio for Nile tilapia (Oreochromis niloticus) breeding females, Aquaculture (2019), doi: https://doi.org/10.1016/ j.aquaculture.2019.734625. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

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Effects of the dietary linoleic acid to linolenic acid ratio for Nile tilapia (Oreochromis niloticus)

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breeding females

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Tamira Maria Orlandoa*, Táfanie Valácio Fontesa, Renan Rosa Paulinoa, Luis David Solis Murgasa,

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Jose Fernando López-Olmedab, Priscila Vieira Rosaa**.

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a

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37200-000, Brasil.

Departamento de Zootecnia, Universidade Federal de Lavras, UFLA, Lavras, Minas Gerais,

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b

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Espanha.

Departamento de Fisiologia, Faculdade de Biologia, Universidade de Murcia, 30100, Murcia,

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*Corresponding author

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**Correspondence to: P.V. Rosa, Departamento de Zootecnia, Universidade Federal de Lavras s/n,

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P.O. Box 3.037, 37200-000 Lavras, Minas Gerais, Brazil.

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E-mail addresses: [email protected] (T.M. Orlando), [email protected] (P.V. Rosa).

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ABSTRACT

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The present study was conducted to evaluate the effect of dietary linoleic acid/ linolenic acid (LA/

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LNA) ratio for Nile tilapia (Oreochromis niloticus) breeding females. Four isonitrogenous (38%

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protein) and isolipidic diets were formulated to contain different dietary LA/ LNA ratios (20.1; 4.5;

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3.9 and 0.7) by increasing of linseed oil at the expense of corn oil. 96 fishes were stocked in twelve

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250-L each tank and their reproductive performance assessed over 105 days. Reproductive and

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growth performances, fatty acid composition, total lipids in eggs, plasma profile of lipoproteins and

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estradiol and expression of steroidogenic genes on ovaries were examined. At the end of the

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experiment, fish fed the 3.9 and 0.7 LA/ LNA diets produced higher number of eggs (p< 0.05). In

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general, the fatty acid profile of ovaries and eggs reflected the composition of the diet. The

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arachidonic acid content was higher in the 20.1 ratio and the eicosapentaenoic (EPA) and

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docosahexaenoic (DHA) acids percentage, higher in the 0.7 ratio (p< 0.05). Diets did not influence

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the triglycerides content in plasma (p> 0.05). However, higher levels of cholesterol were observed

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in fish fed 4.5 and 3.9 LA/ LNA diets (p< 0.05). The highest dietary LA levels increased the gene

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expression of cyp17, while the dietary 3.9 LA/ LNA ratio promoted higher expression of cyp19a1a

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(p< 0.05). In conclusion, this study suggests that lower dietary n-6/ n-3 PUFA ratio for Nile tilapia

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female should be used to highest expression of ovarian aromatase and better spawning performance.

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Keywords: Fishes; Broodstock nutrition; Reproduction; Fatty acids; Steroidogenesis

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1 INTRODUCTION

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For a long time, fish oil was used as the main lipid source in aquafeeds. Because the

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shortage supplies and increasing of prices worldwide (Sprague et al., 2016), the use of vegetable

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oils as a substitute to fish oil is a current issue to allow the formulation of more sustainable and

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cheaper diets. However, the understanding the influence of dietary fatty acids, is fundamental for

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elaboration of adequate artificial diets during the pre-spawning period, which can provide better

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spawning success, higher quality and quantity of eggs, hatchability and quality of larvae (Izquierdo,

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M. S., Fernandez-Palacios, H., & Tacon, 2001).

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Lipids are recognized as the major dietary factor in broodstock nutrition by influencing the

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reproductive success (Bell and Sargent, 2003), and the long chain polyunsaturated fatty acids (LC-

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PUFA) n-3 and n-6 series are pointed as one of the main factors by the improvement in the quality

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of fish eggs and larvae (Izquierdo, M. S., Fernandez-Palacios, H., & Tacon, 2001; Lavens et al.,

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1999; Tocher, 2010). In general, vertebrates cannot synthesize de novo n-3 or n-6 series of 18C-

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polyunsaturated fatty acids (PUFA), such as linoleic (18:2n-6, LA) and linolenic acids (18:3n-3,

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LNA) from oleic acid (18:1n-9), due to the lack of ∆12 and ∆15 desaturases enzymes (Tocher et al.,

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2006). Therefore, these essential fatty acids should be supplied by the diets (Sargent et al., 2002).

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18C-PUFA are converted in their biologically active LC-PUFA, namely arachidonic acid

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(20:4n-6, ARA), eicosapentaeneoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA).

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The ability to conversion varies according to species and it is dependent of the presence and

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expression of fatty acids elongation and desaturation genes (Tocher, 2003). Freshwater fish species

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can convert 18C-PUFA in their respective LC-PUFA, through the activity of the desaturase and

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elongase enzymes. However, n-3 and n-6 PUFA share the same bioconversion pathways and a

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competition between them may occur, compromising LA or LNA metabolism (Glencross, 2009).

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Dietary n-3 and n-6 fatty acids have received attention in several studies of fish

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reproduction (Bell and Sargent, 2003; Bruce et al., 1999; Furuita et al., 2007; Liang et al., 2014).

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ARA and EPA have a major role in the synthesis of eicosanoids, a group of biologically active

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products. ARA is recognized to produce series-2 prostaglandins and leukotrienes, which have more

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potent effects than series-3, produced from EPA. Prostaglandins mediate oocyte maturation process

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and affects steroidogenesis (Ann Sorbera et al., 2001). Furthermore, LC-PUFA modulate gonadal

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steroid synthesis (Ann Sorbera et al., 2001) and may act in the tissues or intracellularly as ligands

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for transcription factors controlling gene expression (Tocher, 2015). Furuita et al. (2007) suggested

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that the inclusion of both dietary n-3 and n-6 fatty acids for reproduction is necessary in Japanese

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eel (Anguilla japonica). Liang et al. (2014) related that dietary fatty acids n-3 and n-6 ratio affected

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the spawning performance, egg and larval quality of tongue sole (Cynoglossus semilaevis).

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Spawning performance and larval quality were better in Atlantic halibut (Hippoglossus

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hippoglossus) fed 1.8% of dietary ARA than fish fed a level of 0.4% (Mazorra et al., 2003).

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Cholesterol transport from the outer to the inner mitochondrial membrane, mediated via the

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steroidogenic acute regulatory protein (StAR), is the main rate limiting step of steroidogenesis

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(Arukwe, 2008). The first reaction in the biosynthetic pathway is the conversion of cholesterol to

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pregnenolone by the cytochrome P450 cholesterol side-chain cleavage (P450scc) (Arukwe, 2008).

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Then, a series of enzymes act in pregnenolone to synthesize steroid hormones such as estrogens,

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which regulate fundamental processes for fish reproduction like vitellogenesis, oocyte maturation

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and ovulation (Yaron and Levavi-Sivan, 2011). The enzyme Cyp19a1a (ovarian aromatase) drives

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the transformation of androgens to estrogens, thus being a key enzyme for the control of ovarian

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physiology and reproduction (Guiguen et al., 2010; Yaron and Levavi-Sivan, 2011).

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Nile tilapia is the second most produced species in the world (after carps) (FAO, 2018). The

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rapid industrialization of tilapia culture became a challenge to elaborate artificial diets and the use

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of alternative lipid sources might be a way to developing sustainable aquafeeds. Requirements of

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LA and LNA are indicate to be around 1% of diet dry weight for juveniles freshwater (NRC, 2011).

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However, more information about nutritional requirements is needed (Furuya, 2010), especially in

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breeders of this species. Nile tilapia females are multiple spawners, with early sexual maturation

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and short vitellogenic period (Izquierdo, M. S., Fernandez-Palacios, H., & Tacon, 2001) with low

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number of eggs produced in each spawning. Furthermore, they are mouth-brooders and demonstrate

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high parental care (Coward and Bromage, 2000). The low fecundity combined with the

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asynchronous spawning are factors that limit the production of quality seeds. Therefore, the present

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study, aimed to evaluate the effects of dietary linoleic/ linolenic ratios on reproductive performance,

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fatty acid profile and gene expression of proteins related to steroidogenesis in Nile tilapia female.

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2 MATERIAL AND METHODS

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2.1 Fish and culture facilities

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All procedures adopted were in accordance with ethical standards and approved by the

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Ethics Committee of Animal Welfare of the Federal University of Lavras, protocol number

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006/2015. The trial was conducted at the fish farming Station of Federal University of Lavras,

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UFLA, Brazil. A total number of 96 Nile tilapia UFLA strain were acclimatized to experimental

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conditions. UFLA strain underwent genetic improvement programs at the fish farming sector of the

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Federal University of Lavras over the last 25 years (Dias et al., 2016). Triplicate groups were

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randomly distributed into 12 tanks (250-L capacity). Each tank was stocked with 6 female (mean

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initial weight, 124.5g) and 2 male (mean initial weight, 131.3g), in a male/ female sex ratio of 1/3

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as recommended by Litlle and Hulata (2000) under aquarium conditions. All fishes were implanted

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with microchips before beginning the experiment. The culture system consisted of an indoor

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thermoregulated recirculation water system supplied with a continue flow of aeration and sand

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filter. Water temperature was kept at 28 ± 0.5°C and the photoperiod was set at 12h light:12h dark

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cycle.

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2.2 Experimental diets

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Four isonitrogenous (38% crude protein), according to Oliveira et al. (2014), and isolipidic (7% lipids) diets were formulated (Table 1).

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The diets were formulated to have different n-6/ n-3 ratio obtained from corn oil (CO) and

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linseed oil (LO), rich in linoleic acid (LA) and linolenic acid (LNA), respectively. The ingredient

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composition of all diets was similar, except the amount of CO and LO, used to provide different n-

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6/ n-3 ratio by increase the percentage of LO and decrease of CO. Since the requirement of the LA/

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LNA ratio for tilapia breeders has not been determined, the requirements found in a recent study

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conducted in our working group for tambaqui (Colossoma macropomum) juveniles (Paulino et al.,

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2018), a freshwater species as well as tilapia, were used as reference to established the different LA/

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LNA ratios in the present work (20.1; 4.5; 3.9 and 0.7).

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Palm oil was added in constant amount to provide short and medium chain fatty acids

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(Table 2). The residual oil from fishmeal was minimal, about 0.74%. The ingredients were finally

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ground and experimental diets were prepared by mixing the ingredients with oil and water. Diets

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were pelletized in an electrical pelletizing machine dried in a forced air flow oven at 40 °C for 48 h

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and stored at -20 °C until use. Experimental diets were hand-fed to apparent visual satiation, twice a

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day (9:00 h and 15:00 h), during 105 days.

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2.3 Reproductive management

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The reproductive state of all female was verified on a daily basis by examining the oral

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cavity to check the presence of eggs. When present, eggs were gently removed from their buccal

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cavity, counted and weighted on a digital precision scale (0.001 g). The weight of females was

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registered before returning them to their respective tank. The following reproductive parameters

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were analyzed:

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Absolute fecundity = mean number of eggs at each spawning per fish.

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Total spawning per fish = total number of spawning/ number of spawned female.

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Total spawning per tank.

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Total eggs spawned per fish = total number of eggs per tank per number of spawned female.

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Hatchability = number of hatched larvae/ number of eggs *100.

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In the first 60 days of experiment, eggs were collected and stored at -20 °C for further fatty

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acids and total lipids analyses. In the remaining time of experiment, the collected eggs were counted

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and submitted to artificial incubation in 3-L capacity plastic incubators with rounded bottom in a

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thermoregulated (28 °C) recirculation system. A gently flow of water was maintained to ensure a

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slow movement of the eggs, as occur on female’s tilapia mouth. Percentage of hatching was

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recorded.

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2.4 Sample collection

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At the end of the experiment, sampling of all females occurred after 24h of fasting. Blood

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was collected from caudal vein with syringe containing EDTA (4%), immediately centrifuged at

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1000g for 10 min, and plasma collected and stored at -80 °C until analysis. Then, fishes were

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euthanized by an overdose of 2-phenoxyethanol solution (1:500 v/v, Fluka; Sigma-Aldrich, Madrid,

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Spain). Fishes were dissected, and the weights of the liver, ovaries and viscera were recorded to

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determine the hepatosomatic index (HSI), gonadosomatic index (GSI), and viscerosomatic index

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(VSI), respectively, as a percentage of the tissue in relation to the body weight. Liver, ovaries and

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muscle samples were collected and stored at -80 °C to further analysis.

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2.5 Chemical analysis

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2.5.6

Proximate analysis

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Chemical composition of diets was analyzed according to AOAC methods (AOAC, 2005).

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Briefly, dry matter content was determined in an oven for 24 h at 105 °C until constant weight. Ash

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was determined by incineration of the organic components for 5 h at 550 °C using a furnace (LF

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0213, JUNG, São Paulo, Brazil); Kjeldahl method was used for the evaluation of crude protein

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content (N×6.25) after acid digestion; crude lipids determined by extraction with ethyl ether in a

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Soxhlet extraction system.

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2.5.7

Fatty acid and total lipids analyses

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Lipid extraction and FA profile of diet, liver, muscle, ovaries, and eggs were analyzed

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according to Araújo et al. (2017). Briefly, total lipid was extracted with chloroform and methanol

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using a modification of the method from Folch et al. (1957) FA profile was analyzed using a

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GC2010 gas chromatograph (GC) (Shimadzu, Kyoto, Japan) equipped with a flame ionization

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detector (FID) and a SP-2560 fused silica capillary column (100.0 m 9 0.25 mm, 0.20 lm film;

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Supelco, Sigma-Aldrich, St. Louis, MO, USA). Fatty acid peaks were integrated using GC solution

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chromatography software (version 4.02), and peaks were identified by comparison to known

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standards (37 Component FAME Mix; Supelco, Sigma-Aldrich). Total lipids of eggs were determined gravimetrically by chloroform/ methanol (2/1)

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extraction, according to Folch et al. (1957).

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Plasma

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Commercial kits were used to perform triglycerides (TRI - Triglycerides Liquiform, Cat.

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87), total cholesterol (CT - Cholesterol Liquiform, Cat. 76). Plasma samples were incubated with

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the respective color reagent, following manufacturer’s recommendations. Tests were made before

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analysis, and no dilution was necessary. Plasma concentration of estradiol was analyzed by a

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commercial ELISA kit (OE – Biomedix, Cat. EIA2693) for human, as it has been demonstrated

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elsewhere to be valid for tilapia plasma samples (de Alba et al., 2019). Plasma was diluted and tests

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were made to obtain the better adjust of the curve. The limit of detection of the kit is 10.6pg/ mL

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and the calculated values of the percentage of coefficients of variability (% CV) were 5.4% and

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10.6% for intra and inter assay, respectively. Samples were quantified in triplicate for all analysis in

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a 96-well plate and read in a spectrophotometer (Multiskan GO, Thermo Scientific, USA).

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2.5.8

Gene expression

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Total RNA was extracted from ovary of each fish using Trizol reagent in sterile tubes,

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according to the manufacturer’s recommendations (Invitrogen, CA, EUA). RNA concentration was

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determined by spectrophotometry (DeNovix DS-11). Complementary DNA (cDNA) synthesis was

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realized with 2µg of the resulting RNA using the High-Capacity RNA-to-cDNA kit (Thermo Fisher

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Scientific), following manufacturer's instructions. cDNA was submitted to real-time quantitative

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PCR (q-PCR) analysis in a light thermocycler (7500 Real-Time PCR system, Applied Biosystems,

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CA, USA). q-PCR reactions were performed using SYBR-green primer master mix (Applied

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Biosystems) in a final volume of 20 µL. All samples were tested in triplicate and every

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amplification experiment also included non-template controls (NTC). Primers for tilapia

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steroidogenic genes were designed with the Primer3 software (Untergasser et al. 2012) using the

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sequence obtained for each gene from the Ensembl database (www.ensembl.org) (Table 3). The

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primers of 18s, βactin, cyp17 and cyp19a1a were added to a final concentration of 200 nM, and the

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cyp11a and hsd3β1 primers were added to a final concentration of 400 nM in the reaction. The

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relative amplification efficiencies of all of the genes were analyzed using cDNA dilution curves and

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primer concentrations were determined using a primer dilution curve. Two housekeeping genes, 18s

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and βactin, were selected after checking that the coefficient of variation (CV) for each gene within

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each tissue was lower than 5%. The relative expression levels of the samples were calculated by the

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2−∆∆Ct method (Livak and Schmittgen, 2001). The first normalization was performed for all samples

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using the geometric mean of the two housekeeping genes (Vandesompele et al., 2002). The second

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normalization in the 2–∆∆CT calculations was performed using the sample with the lowest value

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within each gene as reference.

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2.6 Statistical analysis

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Data were checked for normality and homogeneity of variances by the Shapiro Wilk and

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Levene tests, respectively. Percentage data were transformed to homogenize variance by square-

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root arcsine. Data were submitted to one-way analysis of variance (ANOVA). Significant

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differences among means were determined using Tukey’s HSD test. A probability level of 0.05 was

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used to reject the null hypothesis. Statistical analyses were done using IBM SPSS Statistics for

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Windows, Version 23.0 (Armonk, NY, USA). Data are present as mean ± standard deviations.

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3 RESULTS

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The observed mortality throughout the test was low and it was not affected by the

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experimental diets. Throughout the test, two mortalities were verified, one fish in the 20.1 and 3.9

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treatments each, that jumped out of the thanks.

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3.1 Growth performance

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At the end of experiment, final body weight of female Nile tilapia was not affected by

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dietary treatments (p > 0.05). Fish fed the 0.7 LA/ LNA diet presented the highest specific growth

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rate (p < 0.05). No differences were found on body indexes, except for HSI that was significantly

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higher in fish fed the 4.5 LA/ LNA diet than those fed the 0.7 LA/ LNA diet (Table 4).

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3.2 Reproductive performance

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Reproductive performance data of female Nile tilapia are present in Table 5. The highest

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absolute fecundity was observed in fish fed 3.9 LA/ LNA diet (p < 0.05). No difference was

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observed on total spawning per fish and per tank (p > 0.05). However, fish fed the 3.9 and 0.7 LA/

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LNA diets presented the highest number of eggs spawned per fish (p < 0.05). Egg percentage

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hatchability was not affected by the different diets (p > 0.05).

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3.3 Fatty acid composition in tissues and total lipids on eggs

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The fatty acid profiles of gonad, liver, and muscle differed in response to the experimental

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diets. Fatty acid composition of the gonad was significantly different (p < 0.05) among treatments

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(Table 6). Lipid profile in the ovaries generally reflected the fatty acid composition of the respective

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treatment. Highest levels of total PUFA n-6 were deposited in the ovaries of fish fed the 20.1 LA/

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LNA diet compared to fish fed with the other diets, whereas more PUFA n-3 was observed in the

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ovaries of fish fed the 0.7 LA/ LNA diet. Following this trend, 18:2n-6 percentage was lower gonad

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of fish fed 0.7 LA/ LNA diet, that also had lowest levels of ARA (p < 0.05), due the lowest

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proportion of LA/ LNA. The highest levels of ARA were observed in 20.1 LA/ LNA diet (p < 0.05)

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and highest percentage of EPA and DHA on total fatty acid (TFA) were observed in fish fed 0.7

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LA/ LNA diet. Similar to the ovaries, the fatty acid composition of eggs generally reflected the

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dietary fatty acid composition and it was very similar to the ovaries from which they were

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originated. Fatty acids profile of eggs represent the mean of spawns and they are shown in Table 7.

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In the liver (Table 8), despite the abundance of LA in the 20.1 LA/ LNA diet, its percentage

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was not different among dietary treatments. Similarly, ARA percentage in TFA was also not

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affected by diets in the liver (p > 0.05). Content of n-3 fatty acids (LNA, EPA and DHA) was

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highest in fish fed 0.7 LA/ LNA diet. Fish fed 4.5 LA/ LNA diet had lowest SAFA content in TFA

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in the liver.

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Fish group fed 0.7 LA/ LNA diet showed lowest LA content in the muscle TFA, but it was

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not different among other diets. Any significant difference was observed in ARA percentage in this

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tissue. LNA percentage on muscle lipids was highest in fish fed the 0.7 LA/ LNA diet. EPA

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percentage was significantly higher in fish fed the 0.7 LA/ LNA diet, compared to fish fed 20.1 LA/

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LNA diet. Higher deposition of DHA on muscle was observed in fish fed 0.7 LA/ LNA diet,

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although it was not different of the group fed 3.9 LA/ LNA diet (Table 9).

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3.4 Triglycerides, cholesterol and estradiol assays

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No significant differences were found on plasma triglycerides and estradiol. Total plasma

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cholesterol was higher in fish fed 4.5 and 3.9 LA/ LNA diets than those fed the 20.1 LA/ LNA diet

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(Table 10).

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3.5 Expression of steroidogenic genes

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Data on expression levels of key steroidogenesis-related genes (hsd3β1, cyp11a, cyp17 and

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cyp19a1a) in the gonad of female Nile tilapia fed the experimental diets are present in Figure 1. No

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differences on expression levels of cyp11a and hsd3β1 were observed (p > 0.05). cyp19a1a (ovarian

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aromatase) expression was highest in fish fed diet 3.9 LA/ LNA (p > 0.05), while fish fed diets 20.1

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and 4.5 LA/ LNA showed the highest expression of the cyp17 in the gonad (p < 0.05).

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4 DISCUSSION

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Several studies have highlighted the importance of the dietary n-6 and n-3 ratio in the

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reproductive performance and egg quality in different fish species (Furuita et al., 2007; Mazorra et

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al., 2003; Wilson, 2009). In the present work, the number of total spawning per tank and per fish

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showed no significant differences among treatments, although the observed values for these

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parameters were slightly higher in fish fed 3.9 and 0.7 LA/ LNA diets. On the other hand, fish fed

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3.9 LA/ LNA diet had highest absolute fecundity, while the treatments 3.9 and 0.7 LA/ LNA

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provided a significantly higher number of eggs spawned per fish. The spawning performance values

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observed in this study are generally within the range reported elsewhere in Nile tilapia breeding

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experiments (El-Sayed et al., 2005; Hajizadeh et al., 2008; Ng and Wang, 2011).

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ARA is recognized as the main precursor of eicosanoids in fish cells. Series-2 of

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prostaglandin are formed from ARA via cyclooxygenase pathway and have been shown to be

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important in reproductive process (Bell and Sargent, 2003), concerning to the control of

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steroidogenesis, oocyte maturation and ovulation (Ann Sorbera et al., 2001; Mercure and Van Der

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Kraak, 1996; Patiño et al., 2003). However, EPA competes for the same enzyme system to

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eicosanoids production, that have lower bioactive effect, and thus exerts a modulating influence in

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the synthesis and efficacy of eicosanoid produced from ARA (Bell et al., 1997). Studies with

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different freshwater species have demonstrated that a suitable dietary n-6 and n-3 ratio is necessary

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to better production of eggs (Furuita et al., 2007; Jaya-Ram et al., 2008). Therefore, the ratio of

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ARA/ EPA may be a critical nutritional factor for ovulating. Increasing levels of dietary LNA in the

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current study seems to have been more appropriate to promote a better ARA/ EPA ratio to the

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control of eicosanoid production for the ovulating process.

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In the present study, hatchability rate varied from 72.9 to 79.4%. Despite the lack of

315

significant differences, high hatchability ratios were achieved in all treatments, and they are higher

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compared to values reported by other laboratories with Nile tilapia breeding (El-Sayed et al., 2005;

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Lupatsch et al., 2010; Ng and Wang, 2011; Tsadik and Bart, 2007). Broodstock nutrition has been

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reported to influence the quality and viability of the embryos (Bhujel et al., 2001; Izquierdo, M. S.,

319

Fernandez-Palacios, H., & Tacon, 2001), because it provides exogenous nutrient required for

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gonadal development. Furthermore, egg yolk is the major source of nutrients for embryonic

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development in fish. We consider that the levels of dietary protein and energy adopted in the current

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work were sufficient to meet dietary nutrient requirements of female broodstock and provide good

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embryo performance.

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Fish fed 0.7 LA/ LNA diet had higher specific growth rate than other groups. However, the

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final weight gain was not different. Despite the large difference observed in the growth parameters,

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no statistically significant difference was observed. This may be due to the high variability of the

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fish sizes throughout the trial, which justifies the high standard deviation observed. Female Nile

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tilapia is characterized to be a mouth-brooder and during spawning fish do not eat while they are

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incubating their eggs in the mouth. Tilapia females were kept with males during all experimental

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period, so feed intake could not be quantified. Despite lower body growth rates, parameters such as

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lipid composition and fatty acid profile observed in fish fed 4.5 LA/ LNA diet were only different

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from the 3.9 LA/ LNA group and the hatching rate observed was comparable to the other groups.

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Suggesting that tilapia species can suppress the growth to maintain their reproductive capacity

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(Coward and Bromage, 1999).

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HSI have been used as an indicator of the reproductive period being correlated with GSI,

336

since the liver has an important role in reproduction, regarding expressing estradiol receptors to

337

determine the vitellogenin synthesis. Highest HSI was observed in the fish fed 4.5 LA/ LNA diet,

338

with no difference on GSI or VSI. In contrast, Nile tilapia female fed linseed oil or cod liver oil, an

339

inverse relationship between growth performance and reproduction was observed (Ng and Wang,

340

2011; Santiago and Reyes, 1993).

341

Studies confirm the ability of freshwater fish to elongate and desaturate the precursors LA

342

and LNA in their respective LC-PUFA, namely ARA, EPA and DHA (Agaba et al., 2004).

343

Evidences have been suggested that tilapia and zebrafish express a fatty acid desaturation and

344

elongation pattern in hepatocytes (Jaya-Ram et al., 2008; Tocher et al., 2002). In the present work,

15

345

the lower percentage of 18:2n-6 and 18:3n-3 in tissues as compared to diets combined with higher

346

percentage of LC-PUFA, demonstrate biosynthesis activities to form LC-PUFA. Furthermore,

347

activities of desaturation and elongation in freshwater fish are nutritionally regulated, being higher

348

with elevated availability of precursor for biosynthesis pathway (Francis et al., 2007; Tocher et al.,

349

2002) and during limited dietary intake of LC-PUFA (Bell et al., 2003), as it occurs when fish are

350

fed with vegetable oil sources.

351

The fatty acid composition of fish ovaries and eggs is recognized to be directly influenced

352

by dietary fatty acid composition as well as in muscle (El-Sayed et al., 2005; Furuita et al., 2007;

353

Lewis et al., 2010). In the present study, the fatty acid n-6/ n-3 ratio of fish tissues and eggs

354

generally reflected the respective treatment. Fish group fed 20.1 LA/ LNA diet (26.7% 18:2n-6

355

TFA) presented the highest levels of ARA in the ovaries and eggs compared to the other fish

356

groups, but its percentage was not significantly different in the liver and muscle, suggesting that

357

dietary LA may have contributed to increase the percentage of ARA in TFA in the ovaries and eggs.

358

Highest levels of EPA and DHA were observed in fish fed 0.7 LA/ LNA diet (18.4% 18:3n-

359

3 TFA) in ovaries, liver and eggs, and high percentage of DHA contributed to elevate total LC-

360

PUFA in the liver, ovaries and eggs, but not in the muscle. In some species reduced feed intake was

361

observed during spawning, and muscle may be a relevant source of LC-PUFA in this period

362

(Almansa et al., 2001; Pérez et al., 2007; Rodríguez et al., 2004). Furthermore, the incorporation of

363

lipids from tissues to eggs provide energy substrate to maintain embryonic development throughout

364

gonadal development (Almansa et al., 2001). Higher concentrations of total lipids were obtained in

365

eggs produced by fish fed 3.4 and 0.7 LA/ LNA diets. During vitellogenesis, vitellogenin is

366

incorporated by oocytes and then storage as yolk proteins. Other nutrients, such as lipids and

367

vitamins are also incorporated (Le Menn et al., 2007; Lubzens et al., 2010). Besides the importance

368

of long chain polyunsaturated fatty acids as eicosanoids precursor, the supply of fatty acids to

16

369

oocytes and their storage in yolk, is an essential process in reproduction and embryo quality

370

(Izquierdo, M. S., Fernandez-Palacios, H., & Tacon, 2001).

371

In the current work fish fed 4.5 and 3.9 LA/ LNA diets had highest levels of plasma

372

cholesterol compared to fish fed 20.1 LA/ LNA diet, but no changes in triglycerides were observed.

373

Cholesterol is the common substrate for the synthesis of all steroid hormones and its transfer from

374

the cytoplasm to the inner of mitochondrial membrane is the rate limiting step of steroidogenesis

375

(Manna et al., 2009; Stocco and Clarck, 1996). Although not determined, dietary cholesterol levels

376

probably did not vary, since the only source of cholesterol was from fishmeal, which was added in

377

constant amount in all diets. Therefore, the observed differences in plasmatic cholesterol was due to

378

the endogenous synthesis. Transcription of cholesterol and LC-PUFA biosynthesis genes in fish

379

seems to be mediated by activation sterol regulatory element binding proteins (SREBPs) (Eberlé et

380

al., 2004).

381

Gonadal steroidogenesis regulation occurs mainly through gonadotropins: follicle-

382

stimulating hormone (FSH) and luteinizing hormone (LH) (Lubzens et al., 2010). This regulation is

383

related to the expression of an array of genes that encodes steroidogenic enzymes, which ensure the

384

ability of cells to produce steroids hormones from cholesterol and its derivatives (Guzmán et al.,

385

2014; Ijiri et al., 2003; Kagawa, 2013; Lubzens et al., 2010). The first step for biosynthesis of

386

steroid hormones is the conversion of cholesterol to pregnenolone through the side chain cleavage

387

enzyme (P450scc) (Nagahama and Yamashita, 2008), encoded by cyp11a. Then, pregnenolone is

388

converted to E2 by the action of many steroidogenic enzymes encoded by a number of genes,

389

including hsd3β1 (3β-hydroxysteroid dehydrogenase), cyp17 (17α-hydroxylase and 17,20 lyase)

390

and cyp19a1a (Clelland and Peng, 2009). In this study, experimental diets differentially regulated

391

the gene expression of the key genes related to steroidogenesis. Expression levels of cyp11a and

392

hsd3β1 were not influenced by the diets. However, higher levels of cyp17 were observed in fish fed

393

20.1 and 4.5 LA/ LNA diets.

17

394

17α-hydroxylase and 17,20-lyase activities are required for testosterone synthesis, whereas

395

only 17α-hydroxylase enzyme is required to produce 17α-hydroxyprogesterone, the precursor of the

396

hormone which induce oocyte maturation in teleost (17α,20β-dihydroxy-4-pregnen-3-one, 17α,20β-

397

DHP). A shift on production of E2 to 17α,20β-DHP prior to oocyte maturation was reported in

398

salmonids (Nagahama, 1997; Planas et al., 2000), Japanese eel (Kazeto et al., 2000) and rainbow

399

trout (Sakai et al., 1992), suggesting the cyp17 as a critical factor in the steroidogenic switch. Ings

400

and Van der Kraak (2006) reported decrease of the cyp17 levels with the progress of primary

401

growth to maturating follicles in zebrafish ovary. Results of this work demonstrate that fish from

402

groups 3.9 and 0.7 LA/ LNA had lower expression of cyp17. The lower level of expression of this

403

enzyme and the better reproductive performance observed in these groups throughout the

404

experiment, suggests that may be occurred a downregulation to progesterone synthesis and

405

consequent decrease in the gene expression of cyp17. Because 17α-hydroxyprogesterone is

406

produced from progesterone by the hydroxylase activity of P450c17, its 17,20-lyase activity needs

407

to be downregulated in the postvitellogenic follicles.

408

Highest expression of cyp19a1a occurred in fish fed 3.9 LA/ LNA diet. Expression of

409

cyp19a1a gene has been related in vitellogenic follicles in several species (García-López et al.,

410

2011; Gen et al., 2001; Ijiri et al., 2003; Ings and Van Der Kraak, 2006; Kumar et al., 2000) and its

411

expression coincided with a peak of plasma E2 in rainbow trout (Nakamura et al., 2016). In the

412

present work, despite the differences observed on cyp19a1a, E2 levels were not significantly

413

influenced by the different n-6 and n-3 fatty acid ratios. In addition to stimulating vitellogenesis, E2

414

can stimulate the release of the FSH, important for gametogenesis and steroidogenesis, through

415

positive feedback on the hypothalamic-pituitary-gonadal axis. Although no significant differences

416

in E2 concentrations were observed, a reduction in the 3.9 LA / LNA group was noted. This

417

reduction may have stimulated increased expression of cyp19a1a in this group in an attempt to

418

promote increased levels of this estrogen, important for maintaining the reproductive success.

18

419

In conclusion, the current study demonstrated that dietary n-6/ n-3 ratios influence the

420

spawning performance and the expression of ovarian genes related to steroidogenesis in Nile tilapia.

421

Dietary lipid sources generally reflected fatty acid composition of the tissues. Furthermore, plasma

422

cholesterol levels were influenced by different n-6/ n-3 ratios. Results of this study suggest that

423

dietary LA/ LNA ratio for tilapia should be 3.9 to highest expression of ovarian aromatase and

424

better spawning performance.

425 426

Acknowledgements

427

This work was supported by Council for Scientific and Technological Development

428

(CNPq) and Coordination of Improvement of Higher Level Personnel (CAPES). Jose F. López-

429

Olmeda was funded by a “Ramón y Cajal” research fellowship granted by the Spanish Ministry of

430

Economy and Competitiveness (RYC-2016-20959). We thank the Research Support Foundation of

431

the State of Minas Gerais (FAPEMIG) for the research funding granted to Priscila Vieira Rosa

432

(PPM 00227/12) and the Ph.D. scholarship from CAPES, granted to Tamira Maria Orlando which

433

made possible the accomplishment of this study. We thank the personnel of the Scientific and

434

Technical Research Area (ACTI) of the University of Murcia for their support on qPCR assays. We

435

would also like to thank Eleci Pereira and the Study Center in Aquaculture (NAQUA) staff for their

436

assistance throughout the experiment. We have no conflicts of interest to declare.

437 438 439 440 441 442 443

19

444 445 446 447 448

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unsaturated fatty acid synthesis in marine fish: cloning, functional characterization, and

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671

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672 673 674

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675

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676

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679

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681

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682

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683

12-374553-8.00058-7

684 685 686 687 688 689

30

690 691 692 693 694 695

Table 1 - Ingredient and proximal composition of the experimental diets. Diets

696 697 698 699 700 701 702

Ingredients 20.1 4.5 3.9 0.7 Texturized soy protein1 55.00 55.00 55.00 55.00 Fish meal 8.00 8.00 8.00 8.00 Corn starch 17.00 17.00 17.00 17.00 Gelatin 5.74 5.74 5.74 5.74 Cellulose 2.21 2.21 2.21 2.21 Corn oil 3.00 2.01 1.02 0.00 Linseed oil 0.00 0.99 1.98 3.00 Palm oil 4.00 4.00 4.00 4.00 Dicalcium phosphate2 1.50 1.50 1.50 1.50 Kaolim 2.18 2.18 2.18 2.18 Sodium chloride 0.5 0.5 0.5 0.5 3 0.5 0.5 0.5 0.5 Mineral and Vitamin mix DL-Methionine 0.19 0.19 0.19 0.19 Threonine 0.1 0.1 0.1 0.1 Vitamin C 0.06 0.06 0.06 0.06 Antioxidant BHT 0.02 0.02 0.02 0.02 Proximate composition (% dry weight) Dry Matter 93.87 93.51 94.47 93.16 Crude protein 38.3 38.6 37.9 38.0 Crude lipid 7.4 7.2 7.4 7.3 Ash 9.5 9.3 9.2 9.9 Energy (MJ/ Kg)4 16.73 16.73 16.73 16.73 1 Crude protein 50%, Crude lipid 1.3% 2Nutrimix, MS, Brazil Mix vita/min omnivorous fish Cargill, SP, Brazil. 3Composition (mg kg−1 diet): iron sulfate, 196; copper sulfate, 28; zinc oxide, 280; manganese oxide, 52; sodium selenite, 1.2; cobalt sulfate, 0.4; potassium iodide, 1.2; vitamin A, 19,950 (UI kg−1 diet); vitamin D3, 7980 (UI kg−1 diet); vitamin E, 199; vitamin K3, 10; vitamin C, 700; thiamin, 50; riboflavin, 50; pyridoxine, 50; cyanocobalamin, 0.1; niacin, 200; calcium pantothenate, 100; folic acid, 10; biotin, 1.6; inositol, 100; ethoxyquin, 247. 4Calculated values.

31

703 704 705 706 707 708 709 710

711 712 713 714 715 716 717 718

Table 2 - Fatty acid composition (% of total fatty acids) of the experimental diets. Dietary LA/ LNA ratio Fatty acid 20.1 4.5 3.9 0.7 C14:0 7.06 0.7 7.7 7.7 C16:0 18.3 27.2 12.7 10.5 C16:1 0.5 0.6 0.7 0.6 C17:0 0.0 0.1 0.1 0.1 C18:0 3.3 4.2 3.5 4.3 C18:1n9 25.4 34.5 22.7 21.0 C18:2n6 26.7 24.1 23.2 13.6 C18:3n3 1.3 5.4 5.9 18.4 C20:0 0.3 0.4 0.2 0.1 C20:1n9 0.3 0.3 0.3 0.3 C20:2 0.1 0.1 0.1 0.1 C20:4n6 0.1 0.1 0.2 0.1 C20:5n3 0.6 0.5 0.5 0.5 C22:0 0.1 0.0 0.0 0.1 C22:6n3 0.7 0.7 0.8 0.6 Σ SAFA 29.0 32.7 24.2 22.9 Σ MUFA 26.2 35.4 23.8 22.0 Σ PUFA 29.3 31.3 30.3 33.4 Σ LC-PUFA 1.4 1.3 1.6 1.3 Σ n-6 26.7 24.2 23.4 13.7 Σ n-3 2.6 6.5 7.4 19.6 LA/LNA 20.1 4.5 3.9 0.7 ARA/ EPA 0.2 0.3 0.3 0.2 Σ SAFA: sum of saturated fatty acids; Σ MUFA: sum of monounsaturated fatty acids; Σ PUFA: sum of polyunsaturated fatty acids; LC-PUFA: sum of long chain polyunsaturated fatty acids.

32

719 720 721 722 723 724 725 726

Table 3 - Primer sequences used for quantitative PCR analyses. Gene cyp11a hsd3β1 cyp17 cyp19a1a 18s βactin

727

Fw/Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv

Primer sequence ACCGCGTGATTCTCAACAAG ACAAAATCCTGGCCCACTTC AAGGAACGCAGCTGCTTTTG TCACTTCAATGGTGCTGGTG TGAATTACCACCGCATGTGC ACTTCTTTGGCGTGCACATG TTGCACAAAACCACGGTGAG ACGTGCGGGTTTTGTTTGAG GGACACGGAAAGGATTGACAG GTTCGTTATCGGAATTAACCAGAC TGGTGGGTATGGGTCAGAAAG CTGTTGGCTTTGGGGTTCA

Ensembl/GenBank number ENSONIG00000015391 ENSONIG00000015700 ENSONIG00000009168 ENSONIG00000000155 JF698683 XM_003455949

33

728 729 730 731 732 733 734 735 736 737 738 739 740

Table 4 - Growth performance and body indices of Nile tilapia fed with different LA/ LNA diets for 105 days.

741

Diets Growth performance

20.1

4.5

3.9

0.7

Initial body weight (g)

121.2 ± 4.4

128.1 ± 0.5

124.3 ± 2.0

124.5 ± 4.4

Final body weight (g)

179.1 ± 6.8

169.1 ± 7.6

187.9 ± 11.5

191.4 ± 12.1

Weight gain (g)

58.0 ± 11.2

36.5 ± 7.1

63.7 ± 9.8

63.0 ± 9.2

1

Specific growth rate

0.4 ± 0.1ab

0.3 ± 0.0a

0.4 ± 0.0ab

0.4 ± 0.0b

VSI

2

9.9 ± 0.9

9.6 ± 0.7

9.0 ± 0.3

9.6 ± 0.2

GSI

3

4.4 ± 0.3

3.9 ± 0.4

4.4 ± 0.5

4.3 ± 0.2

HSI

4

1.5 ± 0.3ab

1.7 ± 0.1b

1.6 ± 0.1ab

1.4 ± 0.1a

34

742 743 744 745

Values are mean ± S.D of three tanks. Mean in the same row with different letters are significantly different (p < 0.05) by Tukey’s test. 1Specific growth rate: [(ln final weight - ln initial weight)/ time]*100; 2VSI: viscerosomatic index; 3HIS: hepatosomatic index; 4GSI: gonadosomatic index.

746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761

Table 5 - Reproductive performance female Nile tilapia fed experimental diets for 105 days. Diets

762 763 764 765 766

Reproductive performance

20.1

4.5

3.9

0.7

Absolute fecundity

642.1 ± 73.8a

639.0 ± 85.3a

789.5 ± 74.9b

717.1 ± 109.7ab

Total spawning per fish

4.06 ± 0.25

4.03 ± 0.27

4.33 ± 0.25

4.22 ± 0.35

Total spawning per tank

24.3 ± 1.53

24.1 ± 1.60

26.3 ± 1.53

25.3 ± 2.08

Total eggs spawned per fish

2589.6 ± 75.11a

2683.7 ± 155.94a

3051.1 ± 130.82b

2987.9 ± 203.23b

Hatchability (%)

74.1 ± 11.6

74.3 ± 8.1

79.4 ± 9.5

72.9 ± 10.4

Values are means ± S.D of three tanks. Mean in the same row with different letters are significantly different (p < 0.05).

35

767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784

Table 6 - Gonad fatty acid (% total fatty acids) composition of Nile tilapia fed with different n-6/ n3 fatty acid ratio diets.

Fatty acid C14:0 C16:0 C18:0 C18:1n-9 C18:2n-6 LA C18:3n-3 LNA C20:3n-6 C20:4n-6 ARA C20:5n-3 EPA C22:6n-3 DHA Σ SAFA Σ MUFA Σ PUFA Σ LC-PUFA Σ n-6

20.1 3.9 ± 0.7b 26.6 ± 0.9b 8.0 ± 1.3 27.2 ± 1.1ab 12.4 ± 1.1 b 0.4± 0.1a 1.9 ± 0.2 5.0 ± 0.4c 0.1 ± 0.0a 4.1 ± 0.7a 40.5 ± 1.5b 33.6 ± 1.7b 24.9 ± 1.4a 10.7 ± 1.0a 20.4 ± 0.8c

Dietary LA/ LNA ratio 4.5 3.6 ± 0.2a 25.9 ± 0.8b 7.6 ± 0.8 31.0 ± 1.5b 12.7 ± 0.8b 1.8 ± 0.2b 1.8 ± 0.1 4.2 ± 0.4b 0.2 ± 0.0b 6.0 ± 0.6b 36.2 ± 1.7a 34.5 ± 1.7b 28.4 ± 1.5b 12.0 ± 0.8ab 19.9 ± 0.6b

3.9 3.4 ± 1.2c 23.7 ± 0.4a 8.3 ± 0.6 27.2 ± 1.1ab 11.8 ± 0.9b 1.5 ± 0.1b 1.8 ± 0.1 4.1± 0.4b 0.2 ± 0.0b 6.2 ± 0.9b 41.9 ± 1.7b 30.5 ± 1.1a 26.8 ± 1.6a 12.1 ± 0.8ab 18.6 ± 1.0b

0.7 3.0 ± 1.0c 22.7 ± 1.2a 8.2 ± 0.9 22.7 ± 1.3a 7.6 ± 0.5a 5.0 ± 0.5c 1.8 ± 0.1 2.7 ± 0.2a 0.5 ± 0.1c 8.6 ± 1.1c 41.8 ± 1.3b 30.1 ± 2.3a 27.4 ± 1.8b 12.9 ± 1.4b 12.2 ± 1.6a

36

785 786 787

Σ n-3 4.6 ± 1.3a 8.3 ± 1.1b 8.1 ± 1.1b 14.9 ± 0.6c n-6/n-3 4.6 ± 0.8c 2.4 ± 0.1b 2.3 ± 0.3b 0.8 ± 0.1a Values are means ± S.D of six fish. Mean in the same row with different superscript letters are significantly different (p < 0.05).

788 789 790 791 792 793 794 795 796 797 798 799 800 801

Table 7 - Fatty acid (% total fatty acids) composition and total lipids (% wet weight) in eggs of Nile tilapia fed with different n-6/ n3 fatty acid ratio diets.

Fatty acid C14:0 C16:0 C18:0 C18:1n-9 C18:2n-6 LA C18:3n-3 LNA C20:3n-6 C20:4n-6 ARA C20:5n-3 EPA C22:6n-3 DHA Σ SAFA Σ MUFA Σ PUFA Σ LC-PUFA Σ n-6 Σ n-3

20.1 4.8 ± 0.1b 25.5 ± 0.3b 7.6 ± 0.1b 28.3 ± 0.3b 12.1 ± 1.0b 0.6 ± 0.1a 1.7 ± 0.0c 4.6 ± 0.3c 0.1 ± 0.0a 3.4 ± 0.1a 40.6 ± 0.1b 34.6 ± 0.3b 24.6 ± 1.0a 9.7 ± 0.2a 19.8 ± 1.2b 4.1 ± 0.2a

Dietary LA/ LNA ratio 4.5 2.7 ± 0.1a 25.7 ± 0.2b 7.5 ± 0.1b 29.6 ± 0.2b 12.4 ± 1.1b 1.8 ± 0.2b 1.5 ± 0.0b 3.6 ± 0.1b 0.2 ± 0.0b 5.1 ± 0.3b 37.0 ± 0.1a 35.1 ± 0.2b 27.2 ± 1.1b 10.3 ± 0.3ab 18.9 ± 1.2b 7.2 ± 0.1b

3.9 6.3 ± 0.2c 22.4 ± 0.7a 7.0 ± 0.2a 24.4 ± 1.6a 12.2 ± 0.3b 1.7 ± 0.2b 1.5 ± 0.1bc 3.9 ± 0.3b 0.2 ± 0.0b 5.1 ± 0.3b 39.5 ± 1.4ab 30.4 ± 1.8a 26.2 ± 0.3ab 10.7 ± 0.4b 18.1 ± 0.2b 7.3 ± 0.0b

0.7 6.4 ± 0.8c 22.1 ± 0.1a 7.6 ± 0.0b 23.8 ± 0.0a 8.7 ± 0.0a 5.2 ± 0.0c 1.1 ± 0.1a 2.7 ± 0.0a 0.4 ± 0.0c 7.5 ± 0.2c 40.7 ± 0.8b 30.5 ± 0.1a 28.2 ± 0.3b 11.7 ± 0.2c 13.5 ± 0.0a 13.8 ± 0.2c

37

802 803 804

n-6/n-3 4.8 ± 0.5c 2.6 ± 0.2b 2.5 ± 0.0b 1.0 ± 0.0a Total lipids 10.0 ± 0.3a 9.8 ± 0.7a 12.8 ± 0.1b 12.6 ± 0.5b Values are pooled means ± S.D of three tanks per treatment over consecutive spawns. Mean in the same row with different superscript letters are significantly different (p < 0.05).

805 806 807 808 809 810 811 812 813 814

Table 8 - Liver fatty acid (% total fatty acids) composition of Nile tilapia female fed with different n-6/ n-3 fatty acid ratio diets.

Fatty acid C14:0 C16:0 C18:0 C18:1n-9 C18:2n-6 LA C18:3n-3 LNA C20:3n-6 C20:4n-6 ARA C20:5n-3 EPA C22:6n-3 DHA Σ SAFA Σ MUFA Σ PUFA Σ LC-PUFA Σ n-6

20.1 3.2 ± 0.7 25.1 ± 2.1b 11.2 ± 2.4 27.2 ± 2.5ab 9.3 ± 1.9 0.3 ± 0.2a 1.6 ± 0.3 7.6 ± 2.2 0.1 ± 0.0a 4.7 ± 1.0a 40.6 ± 1.8ab 33.9 ± 3.3ab 24.6 ± 3.9ab 13.9 ± 3.1a 19.6 ± 3.3b

Dietary LA/ LNA ratio 4.5 3.9 3.2 ± 0.3 3.2 ± 1.8 25.0 ± 1.2b 23.0 ± 1.4ab 9.7 ± 1.6 10.9 ± 3.3 31.0 ± 1.7b 27.2 ± 4.6ab 9.6 ± 2.5 9.8 ± 0.9 0.8 ± 0.3b 0.8 ± 0.6b 1.5 ± 0.1 1.5 ± 0.1 6.5 ± 1.8 5.4 ± 2.2 0.2 ± 0.1b 0.2 ± 0.1b 5.4 ± 1.6a 6.1 ± 2.6a 38.0 ± 2.5a 41.8 ± 1.4b 37.4 ± 2.6b 33.3 ± 5.8ab 23.9 ± 4.0a 25.7 ± 4.8ab 12.3 ± 2.8a 13.8 ± 4.1a 17.5 ± 2.8ab 18.5 ± 2.8ab

0.7 2.9 ± 1.5 21.1± 0.8a 12.1 ± 2.7 22.7 ± 3.2a 7.7 ± 1.1 2.8 ± 1.1c 1.5 ± 0.3 5.1 ± 1.4 0.8 ± 0.2c 13.4 ± 1.7b 39.1 ± 1.3ab 28.5 ± 4.1a 30.7 ± 2.8b 21.2 ± 2.6b 15.0 ± 1.2a

38

815 816 817

Σ n-3 5.0 ± 1.0a 6.4 ± 1.6a 7.2 ± 2.5a 15.5 ± 1.7b n-6/n-3 4.0 ± 0.7c 2.7 ± 0.6b 2.5 ± 0.9b 1.1 ± 0.6a Values are means ± S.D of six fish. Mean in the same row with different superscript letters are significantly different (p < 0.05).

818 819 820 821 822 823 824 825 826

Table 9 - Muscle fatty acid (% total fatty acids) composition of Nile tilapia fed with different n-6/ n-

827

3 fatty acid ratio diets.

Fatty acid C14:0 C16:0 C18:0 C18:1n-9 C18:2n-6 LA C18:3n-3 LNA C20:3n-6 C20:4n-6 ARA C20:5n-3 EPA C22:6n-3 DHA Σ SAFA Σ MUFA Σ PUFA Σ LC-PUFA Σ n-6 Σ n-3

20.1 1.8 ± 0.3a 24.6 ± 1.1 9.6 ± 0.6ab 21.4 ±1.4 14.3 ± 0.6b 0.3 ± 0.1a 2.5 ± 0.6 10.7 ± 1.9 0.04 ± 0.1a 3.6 ± 1.0a 38.5 ± 0.8 24.0 ± 1.8 32.3 ± 4.6 19.1 ± 2.0 28.5 ± 3.9c 3.9 ± 1.0a

Dietary LA/ LNA ratio 4.5 1.3 ± 0.4a 24.2 ± 2.0 9.9 ± 1.3b 21.6 ± 2.4 14.2 ± 0.8b 1.0 ± 0.1b 2.2 ± 0.3 9.8 ± 2.1 0.3 ± 0.1ab 5.2 ± 0.5ab 37.2 ± 2.0 24.7 ± 3.2 33.5 ± 1.5 17.0 ± 2.7 27.4 ± 2.3bc 6.3 ± 2.2ab

3.9 5.1 ± 2.8b 22.9 ± 1.6 7.4 ± 1.9a 23.3 ± 1.6 13.9 ± 0.9b 1.6 ± 0.5b 1.8 ± 0.3 6.3 ± 2.6 0.3 ± 0.1ab 6.7 ± 1.0bc 39.5 ± 2.0 28.2 ± 2.2 32.5 ± 2.5 14.9 ± 3.2 23.0 ± 2.7ab 8.8 ± 1.2b

0.7 2.9 ± 1.2ab 23.2 ± 0.9 8.6 ± 1.8ab 22.1 ± 5.1 11.3 ± 0.8a 3.9 ± 0.8c 1.8 ± 0.6 6.2 ± 2.5 0.8 ± 0.3b 8.6 ± 2.9c 37.6 ± 1.2 26.3 ± 3.1 35.1 ± 4.7 18.3 ± 3.8 21.0 ± 3.3a 13.8 ± 2.3c

39

828 829 830

n-6/n-3 7.5 ± 1.5c 5.8 ± 0.5b 2.7 ± 0.6b 1.5 ± 0.0a Values are means ± S.D of six fish. Mean in the same row with different superscript letters are significantly different (p < 0.05).

831 832

Figure Legend

833

Figure 1 - Gene expression of key protein related to steroidogenesis in the gonads of female Nile

834

tilapia fed different LA/ LNA diets. Values are means ± standard error of mean.

835 836

Table 10 - Plasma Nile tilapia parameters fed different LA/ LNA diets for 105 days.

0.7

SEM

220.0b

183.0ab

5.6

256.5

202.6

212.5

9.9

2822.9

2518.5

2748.0

186.8

20.1

4.5

Cholesterol (mg dL )

168.0a

208.9b

Triglycerides (mg dL-1)

243.5

Estradiol (pg mL-1)

2596.0

-1

Diets 3.9

Values are means of three tanks. SEM is pooled of standard error of mean. Mean in the same row with different letters are significantly different (p < 0.05).

HIGHLIGHTS

•

Expression of the genes related to steroidogenesis is differently influenced by the n-6 and n3 fatty acid ratios.

•

Lower dietary linoleic/ linolenic acids ratios increase gene expression of ovarian aromatase.

•

Optimal dietary n-6/ n-3 ratio improves spawning performance in Nile tilapia.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Author contributions

Name of author Tamira M. Orlando; Priscila V. Rosa Tamira M. Orlando; Priscila V. Rosa; Renan R. Paulino Tamira M. Orlando; Priscila V. Rosa; Renan R. Paulino Renan R. Paulino

Tamira M. Orlando; Tafanie V. Fontes; Jose F. López-Olmeda Priscila V. Rosa; Jose F. LópezOlmeda Tamira M. Orlando; Tafanie V. Fontes

Tamira M. Orlando; Priscila V. Rosa

Tamira M. Orlando; Priscila V. Rosa; Luis D.S. Murgas; Jose F. LópezOlmeda Tamira M. Orlando; Priscila V. Rosa; Luis D.S. Murgas; Jose F. LópezOlmeda Priscila V. Rosa

Tamira M. Orlando

Priscila V. Rosa; Jose F. LópezOlmeda

Credit role Conceptualization Ideas; formulation or evolution of overarching research goals and aims Methodology Development or design of methodology; creation of models Validation Verification, whether as a part of the activity or separate, of the overall replication/ reproducibility of results/experiments and other research outputs Formal Analysis Application of statistical, mathematical, computational, or other formal techniques to analyze or synthesize study data Investigation Conducting a research and investigation process, specifically performing the experiments, or data/evidence collection Resources Provision of study materials, reagents, materials, patients, laboratory samples, animals, instrumentation, computing resources, or other analysis tools Data Curation Management activities to annotate (produce metadata), scrub data and maintain research data (including software code, where it is necessary for interpreting the data itself) for initial use and later reuse Writing – Original Draft Preparation, creation and/or presentation of the published work, specifically writing the initial draft (including substantive translation) Writing – Review & Editing Preparation, creation and/or presentation of the published work by those from the original research group, specifically critical review, commentary or revision – including pre-or postpublication stages Visualization Preparation, creation and/or presentation of the published work, specifically visualization/ data presentation Supervision Oversight and leadership responsibility for the research activity planning and execution, including mentorship external to the core team Project Administration Management and coordination responsibility for the research activity planning and execution Funding Acquisition Acquisition of the financial support for the project leading to this publication